Synovial Tissue Of Patients Research Articles

N6‑methyladenosine methyltransferase METTL14 is associated with macrophage polarization in rheumatoid arthritis.

Rheumatoid arthritis (RA) is largely caused by the inflammatory response triggered by macrophage polarization. Through epigenetic reprogramming, the inflammatory state of macrophages can be modified. Macrophage polarization is associated with the RNA epigenetic alteration N6-methyladenosine (m6A) RNA methylation. However, the specific function and underlying mechanisms of m6A methylation in the role of macrophage polarization in RA remain to be elucidated. The mRNA expression levels of m6A methylase genes and signaling pathway components associated with RA macrophages were determined in the present study using reverse-transcription quantitative PCR. Methyltransferase 14 (METTL14) protein expression levels were determined using western blot analysis, and the levels of specific cellular secretion factors were determined using ELISA and flow cytometry. The results of the present study demonstrated that elevated METTL14 expression was associated with joint tenderness, and METTL14 expression was positively correlated with both C-reactive protein and rheumatoid factor expression levels. Moreover, METTL14 exhibited potential in the prediction of visual analogue scale. Pro-inflammatory cytokines (TNF-α) and M1 macrophage markers (CD68+CD86+) were also positively associated with METTL14 expression. The results of the Kyoto Encyclopedia of Genes and Genomes analysis revealed that METTL14 was strongly associated with the MAPK signaling pathway. Notably, JNK and ERK2 exhibited a positive correlation with the M1 macrophage marker, CD68+CD86+, which was positively associated with the pro-inflammatory factor, TNF-α. JNK and ERK2 expression levels were markedly increased in the METTL14 high-expression group, compared with in the low-expression group; however, p38 and ERK1 expression levels were not significantly different between these groups. Collectively, the results of the present study demonstrated that METTL14 expression was significantly increased in the peripheral blood and synovial tissue of patients with RA, highlighting the potential association with both immunoinflammatory markers and clinical symptoms. In addition, it was suggested that METTL14 may exacerbate the downstream inflammatory response, through mediating macrophage polarization via the MAPK pathway.

Mitoregulin modulates inflammation in osteoarthritis: Insights from synovial transcriptomics and cellular studies

Osteoarthritis is a prevalent musculoskeletal disease that involves cartilage degradation, subchondral bone remodeling, and synovial inflammation and ultimately causes physical disability. Common risk factors for osteoarthritis include age, sex, obesity, and genetic predispositions. Treatment includes nonpharmaceutical and pharmacological approaches; however, disease-modifying osteoarthritis drugs remain undeveloped. We aimed to identify key regulatory factors underlying the etiology of osteoarthritis. We studied alterations of the inflammatory responses after manipulating the expression of MTLN, which we selected after sequencing and transcriptomics of the patients’ synovial tissues. MTLN expression was increased in synovial tissues of patients and in SW982 human synovial sarcoma cells following inflammatory stimuli. We found that MTLN overexpression or knockout respectively decreased or increased expression of the inflammation-associated genes, including IL-6, IL-8, and TNF-α. Thus, high levels of MTLN in osteoarthritis may protect tissues against excessive inflammation, thereby offering therapeutic potentials.

Role of CXCL-10 as a Biomarker for Rheumatoid Arthritis

Abstract Background CXCL-10 has been detected within sera, synovial fluid, and synovial tissue in patients with RA and identified as a pro-inflammatory chemokine that mediate leukocyte trafficking and modulate innate and adaptive immune responses. It plays a critical role in the inflammatory response and is involved in several biological processes. Objective To evaluate the predicative significance of chemokine CXCL10 in rheumatoid arthritis (RA) and to focus on its relation to disease activity. Patients and Methods A case control study included 30 patients; were diagnosed with RA, and 30 healthy controls. Clinical examination was done for all patients. Measurement of serum CXCL10 level was done by ELISA, while assessment of disease activity in patients was done using disease activity score (DAS-28). Serum levels of CXCL10 were significantly higher in RA patients than controls. Results Serum level of CXCL-10 was ranging from 349–1380 pg/ml with a mean of 784.6 ± 152.2 in RA patients, which was very high level compared to control group the serum level was ranging from 137 – 476.8 pg/ml with a mean of 338.1 ± 125.5. The difference was statistically highly significant (p < 0.001). AUC for CXCL-10 was 0.951 and the 95% CI was ≤ 1. The cutoff point of CXCL-10 was 450 pg/mL were able to differentiate between RA patients and control group. Most of RA patients (70%) were on methotrexate with dose ranged from 12.5–25 mg per week. Thirty-five (58.3%) patients were on steroids with dose 5-20 mg per day. Comparison of CXCL-10 levels in patients with different grades of disease activity had a statistically significant difference between different grades. Conclusion Our study supports the critical role of CXCL10 in the RA inflammatory cascade. The correlation of CXCL10 with clinical disease activity suggests that CXCL10 plays a central role in RA inflammation and may serve as a disease activity marker in RA. CXCL10 has a significant role as a biomarker for prediction of RA.

SLAMF8 as a potential biomarker for rheumatoid arthritis identified by comparing peripheral blood mononuclear cells, fibroblast‐like synoviocytes, and synovial tissue using bioinformatics analysis

AbstractBackgroundRheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease. Its pathogenesis is not fully understood, and early diagnosis is challenging owing to the lack of effective biomarkers. This study aimed to analyze different samples to identify potential biomarkers and therapeutic targets.MethodsMicroarray datasets of RA, osteoarthritis (OA), and healthy control (HC) were downloaded from the Gene Expression Omnibus database. R software was used to identify differentially expressed genes (DEGs), which were visualized using volcano and heat maps. Venn diagrams, principal component analysis, gene set enrichment analysis, gene ontology, and Kyoto Encyclopedia of genes and genomes were used to analyze the data. A protein–protein interaction network was constructed, and synovial tissues from patients with RA and OA were collected for verification using the collagen‐induced arthritis mouse model.ResultsMore DEGs were found in synovial tissues than in peripheral blood mononuclear cells or fibroblast‐like synoviocytes. Principal component analysis revealed significant differences between the RA and OA samples, highlighting the unique advantages of synovial tissue. Enrichment analysis revealed that metabolic and cytokine signaling pathways play crucial roles in the development of RA. Further analysis of the four synovial datasets identified 54 DEGs, of which signaling lymphocytic activation molecule family (SLAMF) 8 was identified as the key molecule. SLAMF8 levels were increased in the synovial tissue of patients with RA compared to those of patients with OA (0.38 ± 0.19 vs. 12.40 ± 1.66), and SLAMF8 levels were similarly elevated in collagen‐induced arthritis model mice compared with those in the healthy mice (1.13 ± 0.47 vs. 9.05 ± 2.52).ConclusionsThis study established the unique advantages of synovial tissue for RA research and identified metabolic and cytokine signaling pathways as important for RA development. Thus, SLAMF8 may be a potential therapeutic target for RA.

Synovial Matrix Remodeling and Inflammatory Profile in Disc Displacement of the Temporomandibular Joint: An Observational Case-Control Study.

Background: Pain-related temporomandibular joint disorders (TMJD) are a major public health problem, including the diagnoses of disc displacement (DD) with and without reduction (DDwR/DDwoR). Objectives: The study aimed to examine the matrix remodeling and the inflammatory profile in synovial tissues of patients with TMJ-DD, with a view to understand the pathophysiology, and to contribute to the development of tissue-based diagnostic criteria. Methods: This laboratory-based observational case-control study included 30 synovial tissue samples obtained from 30 patients, diagnosed with delayed (DO) or sudden (SO) onset of DDwoR, which were compared against the reference patient material, DDwR (n = 10/diagnosis group). Tissue samples were investigated histologically and via quantitative immunohistochemistry for a panel of antibodies targeted against extracellular matrix proteins and inflammatory markers. The data were analyzed using a generalized linear model with a gamma family distribution (p < 0.05). Results: Quantification of immunostaining revealed significant differences in the distribution of collagen type III (DO, p < 0.001), lumican (DO, p < 0.05), matrix metalloproteinase-2 (DO, p < 0.05), CD4 T-helper cells (DO, p < 0.01; SO, p < 0.001), and CD68 monocytic immune cells (both SO and DO, p < 0.001) in DDwoR groups compared to the reference patient material, DDwR. Conclusions: The observations confirmed differences in matrix remodeling and an increase in local inflammatory activity in the DDwoR diagnosis compared to the reference patient material, DDwR. The study highlighted the importance of synovial tissue characterization to unite micropathology and clinical findings, leading to more reliable diagnostic tools.

The HIF-1/ BNIP3 pathway mediates mitophagy to inhibit the pyroptosis of fibroblast-like synoviocytes in rheumatoid arthritis

BackgroundSynovial hypoxia, a critical pathological characteristic of rheumatoid arthritis (RA), significantly contributes to synovitis and synovial hyperplasia. In response to hypoxic conditions, fibroblast-like synoviocytes (FLS) undergo adaptive changes involving gene expression modulation, with hypoxia-inducible factors (HIF) playing a pivotal role. The regulation of BCL2/adenovirus e1B 19 kDa protein interacting protein 3 (BNIP3) and nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3) expression has been demonstrated to be regulated by HIF-1. The objective of this study was to examine the molecular mechanism that contributes to the aberrant activation of FLS in response to hypoxia. Specifically, the interaction between BNIP3-mediated mitophagy and NLRP3-mediated pyroptosis was conjointly highlighted. MethodsThe research methodology employed Western blot and immunohistochemistry techniques to identify the occurrence of mitophagy in synovial tissue affected by RA. Additionally, the levels of mitophagy under hypoxic conditions were assessed using Western blot, immunofluorescence, quantitative polymerase chain reaction (qPCR), and CUT&Tag assays. Pyroptosis was observed through electron microscopy, fluorescence microscopy, and Western blot analysis. Furthermore, the quantity of reactive oxygen species (ROS) was measured. The silencing of HIF-1α and BNIP3 was achieved through the transfection of short hairpin RNA (shRNA) into cells. ResultsIn the present study, a noteworthy increase in the expression of BNIP3 and LC3B was observed in the synovial tissue of patients with RA. Upon exposure to hypoxia, FLS of RA exhibited BNIP3-mediated mitophagy and NLRP3 inflammasome-mediated pyroptosis. It appears that hypoxia regulates the expression of BNIP3 and NLRP3 through the transcription factor HIF-1. Additionally, the activation of mitophagy has been observed to effectively inhibit hypoxia-induced pyroptosis by reducing the intracellular levels of ROS. Conclusion: In summary, the activation of FLS in RA patients under hypoxic conditions involves both BNIP3-mediated mitophagy and NLRP3 inflammasome-mediated pyroptosis. Additionally, mitophagy can suppress hypoxia-induced FLS pyroptosis by eliminating ROS and inhibiting the HIF-1α/NLRP3 pathway.

Iron overload causes macrophages to produce a pro-inflammatory phenotype in the synovium of hemophiliac arthritis via the acetyl-p53 pathway.

Haemophiliac arthritis (HA) is caused by spontaneous intra-articular hemorrhage and repeated intra-articular hematomas, leading to iron overload, which, in turn, induces M1 macrophage polarisation and inflammatory cytokine secretion, resulting in synovitis. Here, we explored the mechanism by which iron overload in HA induces the polarisation of M1 macrophages, providing a new approach for the treatment of HA synovitis. The synovium from the knee joints of normal amputees and patients with HA was collected. Pathological changes in the synovial tissues were analysed using hematoxylin and eosin staining. Iron tissue deposition was evaluated using the iron assay kit and Prussia Blue staining, while macrophage phenotype was determined using immunofluorescence. The levels of pro-inflammatory cytokines and p53 acetylation were determine using western blotting. An in vitro iron overload model was established by inducing THP-1 macrophages with ferric ammonium citrate, and the involvement of acetylated p53 in M1 macrophage polarisation was investigated. Compared to control samples, the iron content in the synovium of patients with HA was significantly increased. The protein levels of M1 macrophage markers, pro-inflammatory cytokines, and acetylated p53, were also significantly elevated in the synovial tissues of patients with HA. Similar results were observed in the in vitro iron overload model. Furthermore, the inhibition of p53 acetylation in vitro reversed these iron overload-induced effects. In patients with HA, iron overload induced synovial p53 acetylation, leading to macrophage polarisation toward the M1 phenotype and increased inflammatory cytokine secretion, resulting in synovitis. Synovial iron overload is associated with changes in P53 acetylation in hemophiliac arthritis (HA). Acetylated p53, a known regulator of macrophage polarization, is highly expressed in HA synovium, suggesting a potential role in M1 polarization. HA synovial macrophages predominantly polarize into the pro-inflammatory M1 phenotype, secreting elevated levels of pro-inflammatory cytokines.

Noninvasive PET Detection of CD69-Positive Immune Cells Before Signs of Clinical Disease in Inflammatory Arthritis.

Rheumatoid arthritis (RA) is the most common inflammatory joint disease, and early diagnosis is key for effective disease management. CD69 is one of the earliest cell surface markers seen at the surface of activated immune cells, and CD69 is upregulated in synovial tissue in patients with active RA. In this study, we evaluated the performance of a CD69-targeting PET agent, [68Ga]Ga-DOTA-ZCAM241, for early disease detection in a model of inflammatory arthritis. Methods: A model of inflammatory arthritis was induced by transferring splenocytes from KRN T-cell receptor transgenic B6 mice into T-cell-deficient I-Ag7 major histocompatibility complex class II-expressing recipient mice. The mice were examined longitudinally by [68Ga]Ga-DOTA-ZCAM241 PET/CT before and 3, 7, and 12 d after induction of arthritis. Disease progression was monitored by clinical parameters, including measuring body weight and scoring the swelling of the paws. The uptake of [68Ga]Ga-DOTA-ZCAM241 in the paws was analyzed and expressed as SUVmean Tissue biopsy samples were analyzed for CD69 expression by flow cytometry or immunostaining for a histologic correlate. A second group of mice was examined by a nonbinding, size-matched Affibody molecule as the control. Results: Clinical symptoms appeared 5-7 d after induction of arthritis. The uptake of [68Ga]Ga-DOTA-ZCAM241 in the joints was negligible at baseline but increased gradually after disease induction. An elevated PET signal was found on day 3, before the appearance of clinical symptoms. The uptake of [68Ga]Ga-DOTA-ZCAM241 correlated with the clinical score and disease severity. The presence of CD69-positive cells in the joints and lymph nodes was confirmed by flow cytometry and immunostaining. The uptake of the nonbinding tracer that was the negative control also increased gradually with disease progression, although to a lesser extent than with [68Ga]Ga-DOTA-ZCAM241 Conclusion: The uptake of [68Ga]Ga-DOTA-ZCAM241 in the inflamed joints preceded the clinical symptoms in the KRN T-cell transfer model of inflammatory arthritis, in accordance with immunostaining for CD69. [68Ga]Ga-DOTA-ZCAM241 is thus a promising PET imaging marker of activated immune cells in tissue during RA onset.

Clonal Trafficking of Individual T Cells between Peripheral Blood and Inflamed Synovial Tissue in Patients with Immune-Mediated Refractory Arthritis

Background Rheumatoid arthritis (RA) is the most prevalent form of immune mediated arthritis, which is characterised by immune-mediated destruction of synovial joints, leading to synovitis, acute joint pain, reduced motility, and mortality. Enrichment of CD4+ memory T cell subsets with local clonal expansions in the RA synovial tissue (ST) has been extensively reported in the literature. In addition, emerging evidence suggests that CD8+ T cells with inflammatory phenotypes are also present in increased numbers in the ST. However, little is known about whether these populations represent autoantigen-experienced migratory T cells or locally proliferated populations. Here, with the help of paired single cell T cell receptor (scTCR) and RNA sequencing recognizing individual T cell clones, we aimed to gain insights into the trafficking of T cells between peripheral blood (PB) and inflamed target tissue. Methods We collected ST and PB samples from patients with immune mediated arthritis during surgical synovectomy. The patients had been diagnosed with seronegative juvenile arthritis (Pt1-JIA), seropositive RA (Pt2-SP) and seronegative RA (Pt3-SN), and at the time of sampling they were refractory to treatment. We flow sorted T cells and CD45+ leukocytes from ST and PB and profiled them with 10x Genomics 5´-scRNA+V(D)J-seq. Altogether, we captured 68,610 cells passing quality control thresholds. We integrated the data from all individual samples using scVI and visualised the subpopulation structure using UMAP dimensionality reduction as implemented in the Seurat package. Results Altogether we analysed 34134 CD4+ T cells and 23385 CD8+ T cells from PB and ST. In concordance with previous reports, we observed strong transcriptomic differences between the tissue of origin for both CD4+ and CD8+ T cells. We pooled all CD4+ and CD8+ T cells separately and further grouped all cells with identical CDR3b aa sequences into unique clones. In CD8+ T cells, a high number of clones (n=101, pertaining to 7409 cells) was shared between PB and ST. 8 of these clones were enriched to the ST as compared to PB. Interestingly, Pt2-SP harboured a large clone in the ST consisting of 27% of the CD8+ T cells (CDR3b sequence - CASRGGTSITDTQYF). This clone was also detected in PB with a much smaller frequency of only 2% and in a phenotypically different cluster, indicating local proliferation of specific T cell clones in the synovium. By performing a DE gene analysis between cells with the same TCR but between different tissues of origin, we found that the top upregulated genes in ST were CCL4 and CREM suggesting more activated and inflamed phenotype in ST. In the CD4+ compartment, we found only 12 intersecting TCR clones pertaining to 637 cells. Majority of these clones were coming from PB (458 cells from PB and 179 from ST) and were hyperexpanded in PB as compared to ST. No matches for common viral or bacterial epitopes in the existing VDJ database for any of the intersecting clones was found. We then pooled all T cell clones that were enriched in the ST and found 113 clones (p &amp;lt; 0.05, Fisher's exact test). DE gene analysis showed that ST enriched clones have a more activated profile. Pathway enrichment analysis with hypergeometric testing against the GO Biological Process database identified T cell activation and positive regulation of cell adhesion as the top hits. Using GLIPH2, we used publicly available data from a cohort of JIA patients to check TCR level similarity between the two cohorts. We found common motifs between these two cohorts in some of the hyperexpanded clones from PB and ST, and interestingly, TCRs which were predicted to target similar antigens, were enriched to both expanded and intersecting clones. By performing DE gene analysis, we found that predicted TCRs with similarity were more cytotoxic ( GZMH, GNLY, NKG7, GZMB, GZMA) and they overexpressed the TRBV7-6 gene. Conclusions Our results suggest that the cytokine signalling environment in the ST leads to a widespread activation and inflammatory signature within the T cell compartment. This shift in the phenotype occurs within clonal families and is not due to the selective recruitment of activated cells to inflamed tissue. Additional investigations may elucidate the cellular interactions that promote expansion of specific disease promoting clones in ST and provide a more detailed understanding of the spatiotemporal aspects of this process.

Targeting CD44 Receptor Pathways in Degenerative Joint Diseases: Involvement of Proteoglycan-4 (PRG4).

Rheumatoid arthritis (RA), osteoarthritis (OA), and gout are the most prevalent degenerative joint diseases (DJDs). The pathogenesis underlying joint disease in DJDs remains unclear. Considering the severe toxicities reported with anti-inflammatory and disease-modifying agents, there is a clear need to develop new treatments that are specific in their effect while not being associated with significant toxicities. A key feature in the development of joint disease is the overexpression of adhesion molecules, e.g., CD44. Expression of CD44 and its variants in the synovial tissues of patients with DJDs is strongly associated with cartilage damage and appears to be a predicting factor of synovial inflammation in DJDs. Targeting CD44 and its downstream signaling proteins has emerged as a promising therapeutic strategy. PRG4 is a mucinous glycoprotein that binds to the CD44 receptor and is physiologically involved in joint lubrication. PRG4-CD44 is a pivotal regulator of synovial lining cell hemostasis in the joint, where lack of PRG4 expression triggers chronic inflammation and fibrosis, driven by persistent activation of synovial cells. In view of the significance of CD44 in DJD pathogenesis and the potential biological role for PRG4, this review aims to summarize the involvement of PRG4-CD44 signaling in controlling synovitis, synovial hypertrophy, and tissue fibrosis in DJDs.

Exploring CircRNA N6-methyladenosine in human rheumatoid arthritis: Hyper-methylated hsa_circ_0007259 as a potential biomarker and its involvement in the hsa_circ_0007259/hsa_miR-21-5p/STAT3 axis

BackgroundN6-methyladenosine (m6A) is a highly enriched modification found in circular RNAs (CircRNAs); however, the ability and mechanism of CircRNAs to encode for m6A function in rheumatoid arthritis (RA) remain poorly understood. MethodsWe utilized an epitranscriptomic microarray to measure levels and quantities of m6A methylated CircRNAs in synovial tissues of patients with RA and osteoarthritis (OA). We then utilized methylated RNA immunoprecipitation- and MazF-quantitative PCR to identify and validate differentially m6A-methylated RNAs between the groups, conducted a functional enrichment analysis, and selected protein–protein interaction hub genes. Lastly, we predicted and validated the CircRNA/miRNA/mRNA interaction networks. ResultsWe detected 4,845 CircRNAs containing m6A in our samples, with 53 CircRNAs upregulated, and 139 CircRNAs downregulated compared to human OA synovial tissue (|fold change| ≥ 1.2 and p ≤ 0.05). The differentially m6A-modified CircRNAs were associated with the interleukin-6-mediated signaling pathway, with an increase in relative m6A-methylated levels of hsa_circ_0007259 in human RA, a significant decrease in hsa_miR-21-5p, and an increase in signal transducer and activator of transcription 3（STAT3）. The Luciferase Reporter Gene assay verified the binding of hsa_circ_0007259 to hsa_miR-21-5p and the subsequent binding of hsa_miR-21-5p to STAT3. ConclusionWe showed a notable increase in the relative m6A-methylated levels of hsa_circ_0007259 in human RA, indicating a potential role of hypermethylated hsa_circ_0007259 in RA pathogenesis. This may provide valuable insight into the mechanism of RA and the possibility of utilizing hsa_circ_0007259 as a valuable biomarker.

Cytokine-directed cellular cross-talk imprints synovial pathotypes in rheumatoid arthritis

IntroductionStructural reorganisation of the synovium with expansion of fibroblast-like synoviocytes (FLS) and influx of immune cells is a hallmark of rheumatoid arthritis (RA). Activated FLS are increasingly recognised as a...

Pathogenic Role of IL-17 and Therapeutic Targeting of IL-17F in Psoriatic Arthritis and Spondyloarthropathies.

The interleukin 17 (IL-17) family, a subset of cytokines consisting of IL-17A-F, plays crucial roles in host defence against microbial organisms and the development of inflammatory diseases, including psoriasis (PsO), axial spondyloarthritis (axSpA), and psoriatic arthritis (PsA). IL-17A is the signature cytokine produced by T helper 17 (Th17) cells and is considered the most biologically active form. The pathogenetic involvement of IL-17A in these conditions has been confirmed, and its blockade with biological agents has provided a highly effective therapeutical approach. IL-17F is also overexpressed in the skin and synovial tissues of patients with these diseases, and recent studies suggest its involvement in promoting inflammation and tissue damage in axSpA and PsA. The simultaneous targeting of IL-17A and IL-17F by dual inhibitors and bispecific antibodies may improve the management of Pso, PsA, and axSpA, as demonstrated in the pivotal studies of dual specific antibodies such as bimekizumab. The present review focuses on the role of IL-17F and its therapeutic blockade in axSpA and PsA.

Role of high-temperature requirement serine protease A 2 in rheumatoid inflammation

BackgroundHigh-temperature requirement serine protease A 2 (HtrA2) is known to be involved in growth, unfolded protein response to stress, apoptosis, and autophagy. However, whether HtrA2 controls inflammation and immune response remains elusive.MethodsExpression of HtrA2 in the synovial tissue of patients was examined using immunohistochemistry and immunofluorescence staining. Enzyme-linked immunosorbent assay was used to determine the concentrations of HtrA2, interleukin-6 (IL-6), interleukin-8 (IL-8), chemokine (C-C motif) ligand 2 (CCL2), and tumor necrosis factor α (TNFα). Synoviocyte survival was assessed by MTT assay. For the downregulation of HtrA2 transcripts, cells were transfected with HtrA2 siRNA.ResultsWe found that the concentration of HtrA2 was elevated in rheumatoid arthritis (RA) synovial fluid (SF) than in osteoarthritis (OA) SF, and its concentrations were correlated with the number of immune cells in the RA SF. Interestingly, HtrA2 levels in the SF of RA patients were elevated in proportion to synovitis severity and correlated with the expression of proinflammation cytokines and chemokines, such as IL-6, IL-8, and CCL2. In addition, HtrA2 was highly expressed in RA synovium and primary synoviocytes. RA synoviocytes released HtrA2 when stimulated with ER stress inducers. Knockdown of HtrA2 inhibited the IL1β-, TNFα-, and LPS-induced release of proinflammatory cytokines and chemokines by RA synoviocytes.ConclusionHtrA2 is a novel inflammatory mediator and a potential target for the development of an anti-inflammation therapy for RA.

EPCR deficiency ameliorates inflammatory arthritis in mice by suppressing the activation and migration of T cells and dendritic cells.

Endothelial protein C receptor (EPCR) is highly expressed in synovial tissues of patients with RA, but the function of this receptor remains unknown in RA. This study investigated the effect of EPCR on the onset and development of inflammatory arthritis and its underlying mechanisms. CIA was induced in EPCR gene knockout (KO) and matched wild-type (WT) mice. The onset and development of arthritis was monitored clinically and histologically. T cells, dendritic cells (DCs), EPCR and cytokines from EPCR KO and WT mice, RA patients and healthy controls (HCs) were detected by flow cytometry and ELISA. EPCR KO mice displayed >40% lower arthritis incidence and 50% less disease severity than WT mice. EPCR KO mice also had significantly fewer Th1/Th17 cells in synovial tissues with more DCs in circulation. Lymph nodes and synovial CD4 T cells from EPCR KO mice expressed fewer chemokine receptors CXCR3, CXCR5 and CCR6 than WT mice. In vitro, EPCR KO spleen cells contained fewer Th1 and more Th2 and Th17 cells than WT and, in concordance, blocking EPCR in WT cells stimulated Th2 and Th17 cells. DCs generated from EPCR KO bone marrow were less mature and produced less MMP-9. Circulating T cells from RA patients expressed higher levels of EPCR than HC cells; blocking EPCR stimulated Th2 and Treg cells in vitro. Deficiency of EPCR ameliorates arthritis in CIA via inhibition of the activation and migration of pathogenic Th cells and DCs. Targeting EPCR may constitute a novel strategy for future RA treatment.

Exosomal hsa-miR-335-5p and hsa-miR-483-5p are novel biomarkers for rheumatoid arthritis: A development and validation study

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disease that causes cartilage and bone damage. Exosomes are small extracellular vesicles that play a critical role in intercellular communication and various biological processes by serving as vehicles for the transfer of diverse molecules, such as nucleic acids, proteins, and lipids, between cells. The purpose of this study was to develop potential biomarkers for RA in peripheral blood by performing small non-coding RNA (sncRNA) sequencing using circulating exosomes from healthy controls and patients with RA. MethodsIn this study, we investigated extracellular sncRNAs associated with RA in peripheral blood. Using RNA sequencing and differentially expressed sncRNA analysis, we identified a miRNA signature and target genes. Target gene expression was validated via the four GEO datasets. ResultsExosomal RNAs were successfully isolated from the peripheral blood of 13 patients with RA and 10 healthy controls. The hsa-miR-335-5p and hsa-miR-486-5p expression levels were higher in patients with RA than in controls. We identified the SRSF4 gene, which is a common target of hsa-miR-335-5p and hsa-miR-483-5p. As expected, the expression of this gene was found to be decreased in the synovial tissues of patients with RA through external validation. In addition, hsa-miR-335-5p was positively correlated with antiCCP, DAS28ESR, DAS28CRP, and rheumatoid factor. ConclusionsOur results provide strong evidence that circulating exosomal miRNA (hsa-miR-335-5p and hsa-miR-486-5p) and SRSF4 could be valuable biomarkers for RA.

The Identification of Immune-Related Biomarkers for Osteoarthritis Immunotherapy Based on Single-Cell RNA Sequencing Analysis.

Osteoarthritis (OA) is a chronic musculoskeletal disease affecting approximately 500 million people worldwide. Globally, OA is one of the most common and leading causes of disability. Several genetic factors are involved in OA, including inherited genes, genetic susceptibility, and genetic predisposition. As the pathogenesis of OA is unknown, there are almost no effective treatments available to prevent the onset or progression of the disease. In recent years, many researchers focused on bioinformatics analysis to explore new biomarkers for the diagnosis, treatment, and prognosis of human diseases. In this work, we obtain the traditional RNA sequencing data of OA patients from the GEO database. By performing the differentially expressed analysis, we successfully obtain the genes that are closely associated with the OA. In addition, the Venn diagram was applied to evaluate the genes that are involved in OA and immune-related genes. The protein-protein interaction analysis was further conducted to explore the hub genes. The single-cell RNA sequencing analysis was used to evaluate the expression distribution of the MMP, VEGFA, SPI1, and IRF8 in synovial tissues of patients with osteoarthritis. Finally, the GSVA enrichment analysis discovered the potential pathways involved in OA patients. Our analysis provides a new direction for the exploration of the process of OA patients. In addition, VEGFA may be considered a promising biomarker in OA.

Impaired Efferocytosis by Synovial Macrophages in Patients With Knee Osteoarthritis.

Osteoarthritis (OA) exposes all joint tissues to physiologic stresses, increasing the need to clear apoptotic cells from tissues, including the synovium. We undertook this study to assess the burden of apoptotic cells in synovial tissue in patients with late-stage knee OA and to investigate whether OA impairs the macrophage-mediated clearance of apoptotic cells via efferocytosis. Synovial tissue was collected from individuals with healthy knees and patients with late-stage knee OA during arthroplasty. Synovial apoptotic cell burden was assessed by immunofluorescence for cleaved caspase 3. Efferocytosis of apoptotic Jurkat cells by CD14+ synovial tissue macrophages and peripheral blood-derived macrophages was quantified using immunofluorescence microscopy. Effects of OA on macrophage-mediated efferocytosis were modeled by stimulating blood-derived macrophages with synovial fluid collected from individuals with healthy knees and patients with early- or late-stage knee OA. Patients with late-stage knee OA had more apoptotic synovial cells compared to healthy individuals. There was a marked reduction in the fraction of synovial tissue macrophages engaging in efferocytosis and the quantity of material efferocytosed by individual macrophages in OA patients. Blood-derived macrophages exposed to synovial fluid from patients with knee OA recapitulated the defective efferocytosis, with the greatest effect from patients with early-stage knee OA and higher disease activity (pain and inflammation). Apoptotic cells accumulate in the synovium of patients with late-stage knee OA. Our results suggest that OA impairs critical homeostatic functions of synovial macrophages, leading to accumulation of apoptotic cells.

Anti-proliferation and anti-migration effects of Yishen Tongbi decoction in experimental rheumatoid arthritis by suppressing SLC3A2/integrin β3 signaling pathways

BackgroundYishen Tongbi (YSTB) decoction is a patented herbal formula that is used in China to treat rheumatoid arthritis (RA); however, the exact mechanism of its anti-synovial hyperplasia efficacy has not been fully elucidated. PurposeBased on our previous proteomics study, we aimed to reveal whether YSTB inhibits the proliferation and migration of RA-FLSs through the SLC3A2/integrin β3 pathway in vivo and in vitro. Study designThe study design consists of three parts, a comparison of the expression of SLC3A2 and integrin β3 in synovial tissues of RA and OA patients; an animal experiment to verify the pharmacodynamic effect of YSTB, and in vitro experiment to elucidate the specific mechanism of YSTB. MethodsThe expression of SLC3A2 and integrin β3 in the synovial tissues of patients with RA and osteoarthritis (OA) patients were detected by immunohistochemistry (IHC). In vitro, firstly, the proliferation and migration abilities of HFLS (human fibroblast-like synoviocytes) and HFLS-RA (human fibroblast-like synoviocytes-RA) cells were compared by EdU staining and wound healing assays, respectively, and the differences in the expression and localization of SLC3A2, integrin β3, p-FAK and p-Src between HFLS and HFLS-RA cells were detected by IF and WB. In vivo, DBA/1 mice were injected with bovine collagen II to construct a CIA mouse model. Paw swelling, body weight and the arthritis index (AI) were used as basic treatment evaluation indicators for YSTB. Micro-CT and histopathological analyses of the knee and ankle joints were also performed. In addition, the expression of SLC3A2, integrin β3, p-FAK and p-Src in the synovial tissue of mice was detected by IHC. Subsequently, CCK-8 was used to screen for suitable concentrations of YSTB for use in HFLS-RA cells. EdU staining and transwell migration assays were performed to evaluate the inhibitory effect of YSTB on cell proliferation and migration, and WB was conducted to assess whether YSTB inhibited HFLS-RA migration through downregulation of the SLC3A2/integrin β3 pathways. ResultsIHC showed that the expression of SLC3A2 and integrin β3 was higher in RA synovial tissues than in OA tissues. In vivo experiments showed that YSTB inhibited synovial hyperplasia, prevented bone destruction, and reduced the expression of SLC3A2, integrin β3, p-FAK and p-Src. In vitro experiments showed that YSTB inhibited HFLS-RA migration and proliferation by inhibiting the expression of SLC3A2/integrin β3 and downstream signaling molecules. ConclusionYSTB inhibits the proliferation and migration of synovial fibroblasts in RA by downregulating the SLC3A2/integrin β3 pathways.

Identification of CaMK4 as a sex-difference-related gene in knee osteoarthritis.

Osteoarthritis (OA) is a common degenerative joint disease with a higher prevalence in females than in males. Sex may be a key factor affecting the progression of OA. This study aimed to investigate critical sex-difference-related genes in patients with OA and confirm their potential roles in OA regulation. OA datasets GSE12021, GSE55457, and GSE36700 were downloaded from the Gene Expression Omnibus database to screen OA-causing genes that are differentially expressed in the two sexes. Cytoscape was used to construct a protein-protein interaction network and determine hub genes. Synovial tissues of patients (male and female) with OA and female non-OA healthy controls were obtained to confirm the expression of hub genes and screen the key genes among them. Destabilization of the medial meniscus (DMM)-induced OA mice model was established to verify the screened key genes. Hematoxylin and eosin (HE) staining and Safranin O-fast green dye staining were employed to observe synovial inflammation and pathological cartilage status. The abovementioned three datasets were intersected to obtain 99 overlapping differentially expressed genes, of which 77 were upregulated and 22 were downregulated in female patients with OA. The hub genes screened were EGF, AQP4, CDC42, NTRK3, ERBB2, STAT1, and CaMK4. Among them, Ca2+/calmodulin-dependent protein kinase-4 (CaMK4) was identified as a key sex-related gene for OA. It was significantly higher in female OA patients than in the cases of male patients. Moreover, CaMK4 was significantly increased in female patients with OA compared with the female non-OA group. These results suggest that CaMK4 plays an important role in the progression of OA. OA mouse models demonstrated that CaMK4 expression in the mice knee joint synovial tissue elevated after DMM, with aggravated synovial inflammation and significant cartilage damage. Cartilage damage improved after intraperitoneal administration of the CaMK4 inhibitor KN-93. CaMK4 is a key sex-related gene influencing the progression and pathogenesis of OA and may be considered as a new target for OA treatment.